The centromeric histone CenH3 is recruited into the tombusvirus replication organelles

Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting numerous host components and rewiring cellular pathways to build extensive virus-induced replication organelles (VROs) in the cytosol of the infected cells. Most molecular resources are suboptimal in susceptible cells and therefore, tomato bushy stunt virus (TBSV) drives intensive remodeling and subversion of many cellular processes. The authors discovered that the nuclear centromeric CenH3 histone variant (Cse4p in yeast, CENP-A in humans) plays a major role in tombusvirus replication in plants and in the yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV indirectly alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation in situ within VROs by the co-opted glycolytic enzymes to support the energy requirement of virus replication. In summary, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication.

of CenH3 leads to selective re-programming of gene expression of the host, resulting in increased production of glycolytic and fermentation enzymes. These enzymes are exploited by tombusviruses to produce ATP locally within the VROs to achieve robust replication.
In this paper, we studied the role of CenH3 in TBSV replication in yeast, plants and in vitro. Based on knockdown, mutation or over-expression experiments, we showed that CenH3/ Cse4p acts as a cellular restriction factor against TBSV replication. CenH3/Cse4p was found to be partially re-targeted from the nucleus into the cytosolic VROs. In vitro works showed that CenH3/Cse4p binds to the viral RNA and acts as an RNA chaperone. Co-purification and pulldown experiments demonstrated interaction between CenH3/Cse4p and the viral p33 replication protein. However, subsequent analysis showed that TBSV hijacks CenH3/Cse4p into VROs to sequester this histone 3 variant away from the nucleus, which affects the expression of a set of host genes. These genes include pro-tombusviral host factors. We chose to further test the role of CenH3/Cse4p in regulating the glycolytic and fermentation pathways, which are co-opted by tombusviruses. These pathways are usurped by TBSV to provide plentiful ATP within VROs to fuel the activities of additional co-opted host proteins, such as Hsp70, the ESCRT-associated Vps4 AAA ATPase and DEAD-box helicases needed for robust viral replication [41][42][43][44][45][46]. Altogether, CenH3/Cse4p plays both pro-viral and restriction functions during tombusvirus replication.

The nuclear CenH3 histone variant restricts tombusvirus replication in yeast and plants
To explore the possible role of Cse4p (CenH3) in tombusvirus replication, we used the temperature-sensitive haploid yeast strain with a mutated single copy of cse4-1 [17,47]. Partial inhibition of Cse4p by growing the yeast cse4-1 strain at the semi-permissive temperature (32˚C) resulted in a~4-fold increased level of TBSV replicon (rep)RNA [48,49] replication when compared with the BY4741 yeast strain carrying the WT copy of CSE4 (Fig 1A, compare lanes 13-16 to [9][10][11][12]. TBSV replication was also higher in the cse4-1 strain than in the WT strain even at the permissive temperature (23˚C, Fig 1A). This might indicate that a novel activity, not the canonical essential function of Cse4p in chromosome segregation, provides inhibitory effect against TBSV replication. Western blot analysis revealed that the tombusvirus p33 replication protein was expressed close to WT level in the cse4-1 strain (Fig 1A). We also tested the closely-related carnation Italian ringspot virus (CIRV), which replicates on the boundary membranes of mitochondria in contrast with the peroxisome-associated TBSV. CIRV replication is also increased by~4-fold in cse4-1 strain at the semi-permissive temperature ( Fig 1B). These findings suggest that Cse4p is a restriction factor for tombusvirus replication in different subcellular environments.
We used another approach to test the restriction function of CenH3 by expressing the WT Arabidopsis CenH3 in yeast replicating TBSV. We observed~3-fold inhibition of TBSV repRNA accumulation in comparison with the control yeast ( Fig 1C). Similarly, over-expression of the yeast Cse4p also inhibited TBSV repRNA replication in yeast (Fig 1C). These experiments confirmed that under these conditions, CenH3/Cse4 acts as a restriction factor during TBSV replication.
To further explore if the plant CenH3 acts as a restriction factor of tombusvirus replication, we used a virus-induced gene silencing (VIGS) approach to deplete CenH3 level in Nicotiana benthamiana (Fig 1D, bottom panels) [50]. Replication of TBSV genomic (g)RNA was increased by~3-to-5-fold in the CenH3 knockdown plants when compared to the nonsilenced control plants two days after inoculation (Fig 1D, lanes 7-18 versus 1-6). Knockdown of CenH3 (TRV-5'CENH3, Fig 1D) rendered the plants smaller than the control (TRV-cGFP)

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles plants, yet the knockdown plants supported higher levels of TBSV replication, suggesting that low CenH3 expression makes the plants more suited to support TBSV replication. Comparable experiments with CIRV showed that the CenH3 knockdown plants are indeed highly supportive of tombusvirus replication (Fig 1E).
To further test the restriction function of CenH3 against tombusvirus replication in plants, we transiently expressed either NbCenH3 or AtCenH3 in N. benthamiana followed by inoculation of the same leaves with two peroxisome-associated tombusviruses (i.e. TBSV and the closely-related cucumber necrosis virus, CNV) and the mitochondrial membrane-associated CIRV. Northern blot analysis revealed~8-to-10-fold reduction in TBSV, CNV and CIRV gRNA accumulation in the inoculated leaves (Fig 1F-1H). Therefore, all the above data support a strong tombusvirus restriction function of the plant CenH3 histone variant.

Recruitment of the nuclear CenH3 histone variant into the tombusvirus replication organelles in plants
To test if the restriction function of CenH3 is performed in the nucleus or in the cytosol, where tombusviruses assemble the large viral replication organelles (VROs), we co-expressed TBSV p33-BFP replication protein and the GFP-tagged Arabidopsis CenH3, the ortholog of the yeast Cse4, in transgenic N. benthamiana leaves expressing the RFP-H2B (histone H2B) nuclear marker protein (Fig 2A). Confocal laser microscopy analysis revealed the co-localization of p33-BFP and GFP-CenH3 in N. benthamiana cells replicating CNV (Fig 2A). Interestingly, a portion of GFP-CenH3 was still localized in the nucleus marked by the RFP-H2B marker protein in plant cells infected with CNV (Fig 2A). In contrast, GFP-CenH3 was exclusively localized to the nucleus in mock-inoculated plant leaves under these transient expression conditions (Fig 2A, top image). Importantly, the re-localized GFP-CenH3 in the cytosol was present in the TBSV VROs marked by both p33-BFP and RFP-SKL peroxisome luminar marker protein (Fig 2B). The expression of p33-BFP alone (in the absence of TBSV infection) facilitated the partial re-localization of GFP-CenH3 into VRO-like structures (Fig 2B), albeit this process was not as robust as in the case of TBSV or CNV infections. We also performed comparable experiments with the mitochondrial CIRV in either transgenic RFP-H2B or WT N. benthamiana plants. The results showed partial re-localization of GFP-CenH3 into CIRVinduced VRO structures marked by p36-BFP and RFP-CoxIV mitochondrial marker protein ( Fig 2C). The expression of p36-BFP alone (in the absence of CIRV infection) did not induce the re-localization of GFP-CenH3 into VRO-like structures (Fig 2C). Based on these results, we suggest that tombusvirus infections of N. benthamiana plants induce the partial re-localization of the nuclear GFP-CenH3 into the cytosolic VROs.
We also performed subcellular localization experiments in WT yeast cells expressing YFP-Cse4 and co-expressing CFP-p33 together with p92 pol and the repRNA to induce VRO formation [51]. Time point experiments revealed the partial co-localization of YFP-Cse4 with accumulation of TBSV repRNA in yeast expressing A. thaliana His 6 -tagged CenH3 (lanes 1-3) or S. cerevisiae Myc-tagged Cse4p (lanes 7-9) in comparison with the empty vector control (lanes 4-6). Bottom: Western blot analyses of the level of His 6 -p33, His 6 -AtCenH3 with anti-His antibody and Myc-Cse4 with anti-Myc antibody. (D) VIGS-based knockdown of CenH3 expression leads to increased level of TBSV replication in N. benthamiana plants. Top: Northern blot analysis of tombusvirus gRNA and sgRNA accumulation in CenH3-silenced plants inoculated with TBSV. VIGS was performed via agroinfiltration of tobacco rattle virus (TRV) vector carrying 5' or 3'-terminal NbCenH3 sequences or 3'-terminal GFP sequences as control. Middle: Ethidium bromide-stained gel shows ribosomal RNA levels in each sample as a loading control. Bottom: CenH3-silencing restricts the growth of plants. NbCenH3 mRNA levels were analyzed by semi-quantitative RT-PCR in the silenced and control plants. Tubulin mRNA was used as a control. (E) Accumulation of CIRV gRNA and sgRNA in CenH3-silenced N.

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles CFP-p33 at 12 h time point after induction of protein expression (S1 Fig). The co-localization of YFP-Cse4 and CFP-p33 was even more pronounced at the 16 h and 24 h time points (S1 Fig). This is in contrast with the nuclear localization of YFP-Cse4 in WT yeast in the absence of viral components (S1 Fig). Based on these data, we propose that Cse4p is partially re-localized from the nucleus into the cytosolic VROs marked by p33 replication protein in yeast. Altogether, CenH3 and the orthologous Cse4p are re-targeted by TBSV in plant and yeast cells.

The CenH3 histone variant is an RNA chaperone inhibiting tombusvirus replication in vitro
To test if the yeast Cse4 affects TBSV replication in vitro, we reconstituted the tombusvirus replicase by using (+)repRNA transcripts and purified recombinant TBSV p33 and p92 pol replication proteins in cell-free extracts (CFE) prepared from WT yeast ( Fig 3A) [46,52]. The affinity-purified recombinant yeast Cse4p was added in different amounts to the CFE-based replication assay at the beginning of the assay. At the end of the assay, we performed nondenaturing PAGE analysis of the in vitro replicase products. The replication assay revealed up tõ 2-fold reduction in dsRNA replication intermediate and~3-fold reduction in (+)ssRNA products in CFE with the highest amount of Cse4p in comparison with the RNA replication supported by WT CFE in the presence of GST control (Fig 3A, lanes 4-6 versus 1-3). The finding that both the new (-)RNA (present in dsRNA replication intermediate) [53] and the new (+)RNA products were decreased when CFE contained the highest Cse4p level suggests that Cse4p likely inhibits the TBSV replicase assembly steps, which occurs prior to (-)RNA and (+) RNA synthesis in vitro.
To identify the activity of CenH3 important for its viral restriction function, we tested if purified recombinant Cse4p or AtCenH3 could bind to the viral repRNA. Gel mobility shift assays with radiolabeled RNA probes showed that both Cse4p and AtCenH3 bound efficiently to the TBSV (+)RNA template in vitro (Fig 3B and 3C). Template competition assays revealed that three of the four regions of the repRNA with known cis-acting functions during TBSV replication [54][55][56] competed efficiently with (+)repRNA or (-)repRNA templates in vitro (Fig 3D and 3E). Because the secondary structures of these various regions are absolutely critical to support various steps in TBSV replication [57], we tested if CenH3 can modify doublestranded viral RNA structures. We found that Cse4p unwound partial dsRNA regions in the viral repRNA template in the absence of ATP (Fig 3G), whereas Cse4p was not efficient in separating complete dsRNA structure in vitro ( Fig 3F). The ability of Cse4p to bind to the viral RNA and unwind a partial dsRNA template suggests that Cse4p functions as an RNA chaperone in TBSV replication in vitro.
To determine which domain of CenH3 is important to bind to the viral RNA and its chaperone function, we generated a series of truncation mutants of Cse4p, including the N-terminal domain involved in protein interactions and post-translation modifications (protein stability) and the C-terminal Histone-fold domain (HFD) containing the centromere targeting domain (CATD) [58]. Expression of two N-terminal deletion mutants (i. e., ΔN50 and ΔN80) by p33-BFP and RFP-SKL peroxisomal marker protein. Note that a portion of GFP-CenH3 remains in the nucleus. (B) GFP-CenH3 partially re-localizes into the VRO-like structures, in the absence of TBSV replication when only TBSV p33-BFP is expressed. (C) First panel: Co-localization of CIRV p36-BFP replication protein and GFP-CenH3 in cells replicating CIRV. Second and third panels: GFP-CenH3 is re-distributed into the CIRV-induced VROs consisting of aggregated mitochondrial membranes, marked with both p36-BFP and RFP-CoxIV. Fourth panel: GFP-CenH3 does not re-localize into the VRO-like structures, in the absence of CIRV replication when only p36-BFP replication protein is expressed. https://doi.org/10.1371/journal.ppat.1010653.g002

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles To test if CenH3 can also interact with other viral components, we performed co-purification experiments from yeast co-expressing Flag-tagged AtCenH3 and His 6 -tagged p33 replication protein. After detergent-solubilization of the membrane-fraction of yeast, the Flag-CenH3 was immobilized to the Flag-column. Western blot analysis of the eluted proteins from the column revealed the co-purified His 6 -p33 (S3A Fig, lane 1). In a reverse co-purification experiment, we purified Flag-p33 from the detergent-solubilized membrane-fraction of yeast. The purified preparation also contained the Myc-tagged full-length Cse4p (S3B Fig, lane 2). Additional co-purification experiments revealed that the N-terminal fragment of Cse4p was not copurified, whereas the C-terminal HFD domain of Cse4p was present in similarly purified Flag-p33 preparations (S3B Fig, lanes 3 and 4). These co-purification experiments demonstrated the interaction involving p33 replication protein and CenH3/Cse4p in the yeast membrane fraction.
To confirm direct interactions between TBSV p33 and Cse4p proteins in the absence of the viral RNA, we used a pull-down assay with the TBSV GST-tagged p33 and MBP-tagged Cse4p proteins from E. coli (S3C Fig). For the pull-down assay, we used truncated TBSV p33 protein missing its membrane-binding region to aid its solubility in E. coli (termed p33C, S3C Fig).
The GST-based pull-down experiments suggested that the interaction between the p33 replication protein and Cse4p host protein occurs within the highly conserved HFD domain of Cse4p.

Altering host gene expression by TBSV depends on CenH3 in yeast and plants
Previous work with cse4-1 yeast suggested that Cse4p has noncanonical functions in yeast outside of the centromeric area of the chromosome [33,34]. Cse4p acts as a noncanonical regulator of selected number of host genes via replacing histone molecules on the intergenic and promoter regions in chromosomal DNA [33,34]. Interestingly, genes whose expression is affected by Cse4p include several pro-viral host factors needed for robust TBSV replication. These host genes include several glycolytic and ethanol-producing enzymes [33,34], which are also selectively hijacked by TBSV into VROs to provide plentiful ATP locally to promote of purified GST or GST-AtCenH3 along with the 32 P-labeled (+)repRNA or (-)repRNA templates (~0.1 pmol), and unlabeled competitor RNAs (2 and 4 pmol) representing one of the four regions of TBSV DI-72 RNA from both RNA strands (see panel D, top). The GST-AtCenH3-32 P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. (F, G) RNA-strand separation assays. Top: Schematic representation of the RNA/RNA duplexes used in the assays. The templates consist of DI-72 (+)repRNA and a 32 P-labeled complementary (-)RNA creating a complete (F) or partial (G) RNA/RNA duplex. Bottom: Increasing amounts (0.1, 0.2 or 0.4 μM) of purified recombinant GST-Cse4 or GST (as a control), were added to the reactions. The 32 P-labeled RNA products after the in vitro strand separation assay were analyzed on nondenaturing 5% polyacrylamide gels. Three or more independent repeats were performed for each experiment. https://doi.org/10.1371/journal.ppat.1010653.g003

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles efficient TBSV replication [59][60][61]. Indeed, we have confirmed that mRNA expression for pyruvate kinase (PK, termed Cdc19 in yeast), Eno2 (Enolase 2), Pgk1 (phosphoglycerate kinase) and Pdc1 (pyruvate decarboxylase) glycolytic and fermentation enzymes and the proviral Ded1 DEAD-box helicase was increased in cse4-1 yeast at the semi-permissive temperature ( Fig 4A). On the contrary, the expression of SSA1 (Hsp70) and TEF1 (eEF1A) genes has not changed in cse4-1 yeast at the semi-permissive temperature (Fig 4A). Ssa1 and Tef1 are key co-opted host proteins during TBSV replication in yeast [22,45,62,63]. The latter findings suggest that expression of a group of, but not all the pro-viral genes is affected by Cse4p in yeast.
VIGS-based knockdown of CenH3 level in N. benthamiana also led to enhanced expression of Eno2, Pgk1 and Pdc1 glycolytic/fermentation enzymes and the pro-viral RH20 (ortholog of the yeast Ded1) DEAD-box helicase ( Fig 4B). Moreover, we have found that CenH3 knockdown in combination with TBSV infection of N. benthamiana led to the highest expression levels of PK1, Pgk1, GAPC1 (glyceraldehyde-3-phosphate dehydrogenase) and Pdc1 (Fig 4C). TBSV infection did also enhance the expression level of PK1, Pgk1, GAPC1 and Pdc1 bỹ 4-to-8-fold ( Fig 4D). CenH3 expression was increased by~3-fold at 2 dpi, followed by close to normal level of CenH3 expression at 4 dpi ( Fig 4D). On the contrary, the expression of proviral Hsp70-1 and eEF1A plant genes [22,45,62,63] did not change in CenH3 knockdown plants ( Fig 4B). Thus similar to the observations in yeast, the latter findings suggest that expression of a selective group of, but not all the pro-viral genes is affected by CenH3 in plants.
Because the fermentation enzymes (Adh1 and Pdc1) are induced during hypoxia (low O 2 level due to plant submersion in water) by ERF-VII transcription factors in Arabidopsis plants, we measured the expression levels of RAP2.12, HRE1 and HRE2, which are known hypoxia transcription factors [64][65][66]. We have found that CenH3 knockdown in combination with TBSV infection of N. benthamiana led to the highest expression levels of RAP2.12, HRE1 and HRE2, which showed~4-to-9-fold increase ( Fig 4E). Interestingly, CenH3 knockdown or, separately, TBSV infection also enhanced the expression level of RAP2.12, HRE1 and HRE2 ( Fig 4E).
These surprising findings on the shared function of TBSV infection and CenH3-based regulation of expression of glycolytic/fermentation enzymes and hypoxia-related transcription factors led to a new working model. The emerging idea is that TBSV hijacks CenH3/Cse4 from the nucleus into the VROs to affect the normal cellular gene-regulatory function of this conserved histone variant. This leads to alteration of gene expression of selected group of host genes whose expression is affected (directly or indirectly) by the noncanonical function of CenH3/Cse4.

Regulation of expression of selected glycolytic and fermentation enzymes via CenH3 affects local ATP generation within TBSV VROs in yeast and plants
To test the above model, we decided to measure ATP generation in situ within VROs, which depends on the availability of co-opted glycolytic and fermentation enzymes [67]. The ATP level within VROs was measured using a FRET-based biosensor [68], which we have used previously [60,61]. Briefly, we expressed p33-ATeam fusion protein in N. benthamiana leaves. The ATeam domain of the fusion protein can measure ATP level due to a conformational change in the enhanced ε subunit of the bacterial F 0 F 1 -ATP synthase upon ATP binding [60,61]. This is based on increased FRET signal in confocal laser microscopy when the ε subunit binds to ATP, resulting in a conformational change, which results in drawing the CFP and YFP fluorescent tags to close vicinity. On the other hand, the ATP-free form of the ε

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles subunit is present in an extended conformation, which places CFP and YFP fluorescent tags in a distal position. This leads to low FRET signal [68]. We documented previously [60,61] that the p33-ATeam localizes to VROs representing aggregated peroxisomes. We found that the ATP level within VROs was~40% higher in CenH3-silenced plants than in the control nonsilenced N. benthamiana plants (Fig 5A). Similar increased level of ATP production was detected within the CIRV-induced VROs using p36-ATeam fusion protein in CenH3-silenced N. benthamiana plants (Fig 5B). On the contrary, transient over-expression of CenH3 in N. benthamiana leaves reduced the ATP levels within CNV or CIRV-induced VROs by~2-and 4-fold, respectively (Fig 5C and 5D). Overall, the obtained data support the model that CenH3 is a critical host factor affecting the local ATP generation within the tombusvirus VROs in plant cells.
The effect of the orthologous Cse4p was also confirmed in yeast cells by using ATeam-p92 pol biosensor. We found previously [60,61] that the ATeam-tagged p92 pol is a fully functional RdRp, which localizes to VROs representing aggregated peroxisomes in yeast cells.
Since these experiments are best performed in the presence of glucose in yeast medium [61], we used cse4-1 temperature sensitive mutant at semi-permissive 32˚C in comparison with WT BY4741 strain. We detected increased production of ATP within VROs in cse4-1 strain in comparison with the WT yeast strain under the same growth conditions (S4 Fig). The emerging picture from the above experiments is that subversion of CenH3/Cse4p into VROs indirectly facilitates the more efficient ATP generation locally to support the energy requirement of virus replication.

Discussion
Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting the cellular translation machinery, subverting host proteins, intracellular membranes, metabolites and energy to build virus-induced extensive VROs in infected cells. It seems, however, that the molecular resources available in susceptible cells to support robust TBSV replication are suboptimal at the start of viral replication. Therefore, TBSV drives intensive remodeling and subversion of many cellular processes [42,69,70]. The virus-induced changes also include the dramatic alteration of gene expression in the nucleus. How the cytosolic TBSV accomplishes this feat is incompletely understood. The identification of the key role of the centromeric H3 variant in regulation of TBSV replication opens up a new page in TBSV-host interactions as discussed below.

Is the nuclear CenH3/Cse4 histone variant a conventional viral restriction factor for the cytosolic tombusviruses?
Our gene and protein interaction network studies based on a dozen genome-and proteomewide screens, which previously identified host components affecting TBSV replication and recombination or interactions with host components, revealed that Cse4 H3 histone variant is one of the highest connected nodes in the network [18]. Because Cse4/CenH3 (and the orthologous human CENP-A) is a nuclear protein with function within the centromere, it's a puzzle how CenH3/Cse4 could be an important host factor for the cytosolic TBSV. However, yeast studies with a temperature-sensitive mutant of Cse4, over-expression or knockdown of the orthologous CenH3 in plants, all confirmed a strong restriction factor role for CenH3/Cse4 in tombusvirus replication. This conclusion with the peroxisome-associated TBSV is further supported by results obtained with the mitochondria-associated CIRV in yeast and plants.
How can a nuclear, DNA-binding histone variant be a restriction factor for a cytosolic RNA virus? Subcellular localization studies confirmed that CenH3/Cse4 is partially relocalized

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles to the cytosol, namely into the large VROs, during TBSV or CIRV replication. Moreover, CenH3/Cse4 binds to the viral RNA in vitro via its HFD domain. We also showed RNA chaperone activity for Cse4 in vitro, which activity might contribute to the inhibitory function of Cse4 via unwinding critical cis-acting elements in the viral (+)RNA. We also documented interaction of CenH3/Cse4 with the p33 replication protein, which completely overlaps with the N-terminal region of the p92 RdRp. All these results agree with a proposed antiviral activity of the CenH3/Cse4. Accordingly, we have shown in this paper that the purified recombinant Cse4p is indeed inhibitory to TBSV replication in an in vitro replicase reconstitution assay.

Recruitment of CenH3/Cse4 into VROs leads to selective reprogramming of host gene transcription during tombusvirus replication
Our initial results in yeast, plants and in vitro were not consistent with the idea that the canonical function of CenH3/Cse4 is exploited by the host to fight off tombusvirus infection. Moreover, tombusviruses replicate in mature plant leaf and root cells, which are not going through cell division and chromosomal segregation. An interesting noncanonical function of Cse4 is to replace histones bound to the chromosome in many places, including transcription factor hotspots [33][34][35][36]. In this role, Cse4 acts as a negative regulator of gene expression of over a hundred yeast genes [33-35]. Because these genes also include critical host factors, such as glycolytic enzymes, with pro-tombusvirus functions, we propose the idea that tombusviruses subvert CenH3/Cse4 into VROs to alter the gene expression in the nucleus. We propose that the TBSV-driven subversion of CenH3/Cse4 and partial sequestration into VROs have two major consequences for tombusvirus replication: (i) Indirectly altering host gene transcription in the nucleus. Accordingly, we show that similar to knocking down/inhibiting CenH3/Cse4 activities, TBSV infection also increased the expression of selected host genes, namely glycolytic and fermentation enzymes and others. This in turn helps TBSV to recruit pro-viral host factors from the more abundant protein pool, which is the consequence of sequestered CenH3/Cse4 on select gene expression. Altogether, these activities led to increased generation of ATP locally within VROs (Fig 5) likely due to the efficient recruitment of glycolytic and fermentation enzymes. In situ production of ATP within VROs is essential for robust TBSV replication as shown previously [61,67]. On the other hand, over-expression of CenH3/Cse4 might interfere with the TBSV-driven efficient sequestration of this host factor from the nucleus. Indeed, over-expression of CenH3/Cse4 inhibited local ATP generation within VROs and strongly inhibited TBSV replication. (ii) The second direct consequence of subverting CenH3/ Cse4 to the VROs from the nucleus is that this process is "costly" to TBSV. This is because the virus must dedicate viral components, namely portion of the viral (+)RNA population and p33 molecules to subvert CenH3/Cse4 from the nucleus. A lower level of CenH3/Cse4 in the cell via mutation or depletion might help TBSV commit less of its components to sequester CenH3/Cse4 away from the nucleus. Accordingly, depletion or mutation in CenH3/Cse4 led to highly enhanced TBSV and CIRV replication in yeast and plant cells. On the contrary, over-

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles expression of CenH3/Cse4 would force TBSV to commit even more viral components for sequestration (i.e., taking the viral (+)RNA and p33 away from replication functions), thus leading to reduced viral replication. We propose that this sequestration process of CenH3/ Cse4 via TBSV components renders CenH3/Cse4 functioning as a restriction factor under given circumstances in N. benthamiana and yeast. Albeit CenH3 acts as a restriction factor of TBSV replication in N. benthamiana and yeast, one can imagine that CenH3 might act as proviral in other hosts. However, demonstration of this scenario requires future studies.
Hijacking and regulating CenH3 function might be conducted by other viruses as well. For example, Hepatitis B virus x protein (HBx) induces hepatocellular carcinoma (HCC) by inducing the over-expression of CENP-A (CenH3) protein [71]. The hepatitis C virus (HCV)-related chronic liver disease also correlates with increased level of CENP-A expression [72]. Interestingly, the NS1 protein of the influenza A H3N2 subtype contains a histone H3-like sequence (mimicking H3 structure), which is used to hijack host proteins [73].

Summary
CenH3/Cse4 histone variant plays a major role in tombusvirus replication in plants and in yeast model host. The emerging theme from our current studies is that sequestration of CenH3/Cse4 from the nucleus into the cytosolic VROs by tombusviruses requires a balancing act between restriction and pro-viral activities. Robust TBSV replication depends on the virus' ability to reprogram host gene transcription, in which CenH3/Cse4 plays a mostly unexplored role. Subversion of CenH3/Cse4 is a "double-edge sword": possibly advantageous for TBSV under given conditions, but disadvantageous under over-expression conditions, when CenH3/ Cse4 acts as a strong restriction factor. Our data indicate that the nuclear CenH3 histone variant plays surprising novel pro-viral and restriction functions during tombusvirus replication. Nevertheless, the results point at a new frontier in cytosolic tombusvirus-host interactions.

Yeast strains and expression plasmids
Saccharomyces cerevisiae strains BY4741 and R1158 (wt) [74] were obtained from Open Biosystems. See further details in the supplementary materials (S1 Text).

Analysis of tombusvirus replication in yeast
For measuring TBSV repRNA accumulation, yeast strains BY4741 and cse4-1 were transformed with HpGBK-CUP1-Hisp-33/ADH1-DI-72 and LpGAD-CUP1-His-p92. Two sets of cultures per strain were grown at 23˚C 12 h overnight in SC-LH -(synthetic complete medium without leucine and histidine) medium containing 2% glucose and 100 μM BCS (bathocuproinedisulfonic acid). Then, cells were centrifuged and washed thoroughly with clean SC-LHsupplemented with 2% glucose medium, to remove BCS, and pellets were re-suspended in the same medium containing 50 μM CuSO 4 to induce viral protein expression and repRNA replication. Then, a set of cultures was placed at 32˚C and the other set remained at 23˚C and grown for additional 24 h, time after which, total RNA and protein was extracted. For CIRV repRNA accumulation cells were co-transformed with plasmids HpESC-GAL1-Hisp36/ GAL10-DI-72 and LpESC-CUP1-Flag-CIRVp95 and grown as above with the exception that pellets were thoroughly washed with SC-LHmedium (plus 2% galactose) and re-suspended in SC-LHmedium containing 2% galactose and 50 μM CuSO 4 . Total RNA and protein was extracted after 30 h of viral induction.
To over-express CenH3, N. benthamiana leaves were co-infiltrated with Agrobacterium cultures containing pGD-p19 and either pGD empty vector, pGD-NbCENH3 or pGD-AtCENH3. In the experiment with CNV, plants were also infiltrated with Agrobacterium carrying pGD-CNV 20KSTOP 24 h after the first agroinfiltration. In the experiment with TBSV and CIRV, plants were inoculated with crude sap inoculum 48 h after agroinfiltration. Samples were collected from CNV infiltrated leaves about 84 h after the second agroinfiltration. For TBSV and CIRV infection, samples were taken from inoculated leaves 48 h and 72 h post-virus inoculation, respectively. All samples were used for total RNA extraction and northern blot as described, to analyze the accumulation levels of these viruses [76].

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles cultures were grown 12 h overnight in the proper SC-ULHsupplemented with 2% galactose and 100 μM BCS. Next morning, cells were centrifuged and washed (with sterile water) and pellets were re-suspended in the same medium supplemented with 2% galactose and 50 μM CuSO 4 to induce the expression of the fluorescently-tagged proteins. Samples were collected at the time points given in the main figure and analyzed by confocal microscopy as previously described [78].

Estimation of mRNA expression levels for selected host factors
For the mRNA detection in yeast, BY4741 and cse4-1 cells were grown for 12 h at 32˚C. Total RNA was isolated and analyzed by gel electrophoresis in a 1.5% agarose gel to normalize total RNA levels in the samples. Then, the same amounts of total RNA were used for semi-quantitative RT-PCR reactions. cDNA was first obtained using MMLV reverse transcriptase (Lucigen) and oligo dT. The cDNA was then used to perform PCR to detect the expression levels of the following host factors with the following primer sets: #5992/#7136 for Cdc19; #7123/#7137 for Eno2; #6275/#6367 for Pgk1; #5621/#5604 for Pdc1; #4308/#7140 for Ded1; #2030/#7138 for Ssa1 and #7141/#7142 for Tef1.
Real-Time quantitative qRT-PCR was also used for the detection of N. benthamiana gene expression as follows: VIGS was performed as described using the pTRV2-3'CENH3-3' construct only. After 11 d of VIGS treatment, plants were inoculated with TBSV virions or mock inoculated. Samples were collected 2 d post-inoculation from infected leaves and 4 d postinoculation from systemically-infected leaves. Total RNA was isolated and used for qRT-PCR. First, primers were designed using Real Time qPCR Tool from Integrated DNA Technologies website (https://www.idtdna.com/scitools/Applications/RealTimePCR/). Second, MMLV reverse transcriptase (Lucigen) and Oligo-dT were used to obtain cDNA. Finally, the qPCR reactions were prepared using Applied Biosystem Power UP SYBR green master mix (Thermo Fisher Scientific) in a 96 well plate and the Eppendorf's Mastercycler ep realplex instrument and primers #8217/#8218 for GapC1; #8219/#8220 for Pgk1; #8221/#8222 for CenH3; #8174/ #8175 for Pdc1; #8176/#8177 for PK1 and #8178/#8179 for Tubulin β2 as the housekeeping gene control. qPCR conditions were selected following the Power Up SYBR green master mix user manual recommendations.

Visualization and measurement of ATP levels within VROs in plants
Intracellular ATP levels within VROs were visualized using the ATeam-based biosensor [68] by using a confocal microscope and measured by FRET analysis. To detect the ATP levels within the tombusvirus VROs in CenH3-silenced or control N. benthamiana plants, leaves were coagroinfiltrated with plasmids pGD-p33-ATeam YEMK , pGD-p92, pGD-DI-72 and pGD-p19 for CNV infection or plasmids pGD-p36-ATeam YEMK , pGD-p95, pGD-DI-72 and pGD-p19 for CIRV infection. Samples were analyzed in a confocal microscope 2 d post-agroinfiltration. FRET images were obtained by exciting cells with a 405-nm laser diode, and CFP and YFP (Venus) signals were detected at 480-500 nm and 515-615 nm wavelength ranges, respectively [61]. Each YFP/CFP ratio was calculated by dividing pixel-by-pixel a Venus image with a CFP image using Olympus FLUOVIEW software and ImageJ software. In the case of CenH3 over-

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles expression experiments, N. benthamiana leaves were co-infiltrated with the above plasmid combinations for CNV and CIRV with the addition of pGD-NbCENH3 plasmid as well. Confocal FRET images were obtained as above, 2.5 d post-agroinfiltration.  1-2). Expression of ΔN129, ΔC60, ΔC100 and Histone H3 did not affect TBSV repRNA accumulation (lanes 9-16). Middle: Northern blot with 18S ribosomal RNA specific probe was used as a loading control. Bottom: Western blot analyses of the level of His 6 -p33 replication protein and His 6 -Cse4 mutants with anti-His antibody. Note that levels of ΔN129 are very low, suggesting that the N-terminal region of Cse4 has a role in protein stability. (C, D) RNA gel mobility shift analysis shows that GST-Cse4, GST-ΔN50 and GST-ΔN80 efficiently bind to 32 P-labeled (+)repRNA (C) or (-) repRNA (D) in vitro, whereas GST-ΔC100 show defective binding capability to both repRNAs. Purified GST-Cse4, GST-ΔN50, GST-ΔN80, GST-ΔC100 and GST were added in increasing concentrations (0.1, 0.2 or 0.4 μM) to the assays. The 32 P-labeled ssRNA-protein complexes were visualized on nondenaturing 5% polyacrylamide gels. (E, F) RNA-strand separation assays. Left: Schematic representation of the RNA/RNA duplexes used in the assays. See details in Fig  3F and 3G. Right: Increasing amounts (0.1, 0.2 or 0.4 μM) of purified recombinant GST-Cse4, GST-ΔN50, GST-ΔN80, GST-ΔC100 and GST (as a control), were added to the assays. The 32 Plabeled RNA products after the in vitro RNA-strand separation assay were analyzed on nondenaturing 5% polyacrylamide gels. Full length Cse4 and all mutants were unable to unwind a fully duplexed dsRNA (E), whereas WT Cse4 and the N-terminal deletion mutants unwound the partial dsRNA (F). Three independent repeats were performed for each experiment.

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles with Flag-affinity purified Flag-p33 from WT yeast membrane fraction. Myc-Cse4 and Myc-Cse4-CTD were detected with anti-Myc antibody. The negative control was His 6 -tagged p33 (lane 1). Second panels: Western blots of purified Flag-p33 detected with anti-Flag antibody. Bottom panels: Western blot of Myc-Cse4, Myc-Cse4-CTD, Myc-Cse4 N terminal domain (Myc-Cse4-N) and Flag-tagged p33 in the total yeast extracts. Note that after affinity-purification, Myc-Cse4-N was not co-purified with p33 (lane 3). (C) Pull-down assay including TBSV GST-p33 replication protein and the MBP-tagged Cse4 or Cse4 deletion mutants. The C-terminal region of TBSV p33 replication protein was used instead of the full-length protein, which includes the non-soluble N-terminal region with the trans-membrane domains. Top: Western blot analysis of the captured GST-p33C with MBP purified WT GST-Cse4 or ΔN50, ΔN80, ΔN129 Cse4 deletion mutants. GST was used as a control. Note that similar to Myc-Cse4-N, the C-terminal deletion mutants ΔC60 and ΔC100 were not pulled-down with GST-p33C suggesting that the HFD of Cse4 is also important for the interaction between p33 replication protein and Cse4p. Bottom: Coomassie-blue stained SDS-PAGE of the purified recombinant proteins. All experiments were performed three times. (TIF)

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The centromeric histone CenH3 is recruited into the tombusvirus replication organelles